The goal of our grant is to understand the molecular mechanisms by which the lipid second messengers phosphoinositol(4,5)bisphosphate (PIP2) and phosphoinositol(3,4,5)trisphosphate (PIPS) cooperate with small GTPases to promote the axonal-dendritic polarization of neurons. We developed a quantitative working model for axonal-dendritic polarization that involves PIPS and Ras family small GTPases and we will test this model using primary cultured rat hippocampal neurons as a model system. These neurons form a single axon from an initially unpolarized precursor cell and can be used as a prototype neuronal self-polarization process. Our proposed study builds on advances that we made during the last funding period: We used a genomic approach to characterize how small GTPases induce morphology changes (Heo et a!., 2003) and how they are targeted to the plasma membrane (Fivaz et al., 2005;Heo et al., 2006). We also discovered a "geometric attraction" principles that causes a delayed enhancement of plasma membrane targeted proteins in neurites (Craske et al., 2005). We further showed that local PIPS signals are linked to local lamellipod extension, providing a driving mechanism for growth cone extension (Arrieumerlou et al, 2005). In order to overcome a key technical limitation, we developed a chemically-induced enzyme activation method that allows us to rapidly manipulate PIP2 and PIPS lipids as well as small GTPases in the proposed study (Inoue et al., 2005, Shu et al., 2006;Heo et al., 2006). Finally, using a theoretical approach, we found evidence that robust cell polarization requires two interlinked positive feedback loops (Brandman et al., 2005). Specifically, our project makes use of site-directed mutagenesis, live-cell tracking of signaling proteins as well as fluorescence resonance energy transfer measurements to investigate PIP2, PIPS and small GTPase signaling during neuronal polarization. The results form our proposed study will increase our understanding of the molecular mechanisms by which phosphoinositides reversibly target signaling proteins with polybasic clusters and pleckstrin homology (PH)-domains to the plasma membrane and how PIPS signals and small GTPases become polarized when a single axons is specified. Using experimental and quantitative modeling approaches, we will focus on two processes that we discovered: a growth cone restricted positive feedback between HRas and PIPS as well as a directed transport process that enriches HRas in growth cones over the somatodendritic region. We will bring these results together to generate a comprehensive quantitative model of the neuronal polarization process. Neuronal polarization is a fundamental process in the conversion of unpolarized precursor cells to functioning neurons and an understanding of how neurons reliably generate a single axon will provide insights that may advance the development of cures for neurodegenerative diseases.